Anion Exchange Mechanisms in Bacteria

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9603997 Maloney This project concerns Oxlt, a novel transport protein found in the Gram-negative anaerobe, Oxalobacter formigenes. OxlT catalyzed the exchange of external divalent oxalate for internal monovalent formate. This electrogenic exchange, along with proton consumption during intracellular decarboxylation, constitutes an "indirect proton pump" that generates a proton-motive force allowing O. formigenes to extract energy from oxalate. It is now thought such protonmotive metabolic cycles are widely spread in microbiology, and to understand these events at a molecular level this project concentrates on studies on OxlT, the antiport protein at the center of this process in O. formigenes. Antibodies against the OxlT N-terminus are available, the gene is cloned, and functional protein is purified after expression in Escherichia coli. Therefore, tools are available to study structure-function relationships in four areas of progressively increasing resolution: (1) Initial studies will focus on studies of OxlT topology, using peptidespecific antibodies, gene fusions and SH-directed probes, guided by predictions derived from hydropathy analysis of the amino acid sequence. (2) Site-directed mutagenesis will then be used to generate a panel of single-cysteine derivatives to be probed with impermeant SH-active reagents in order to localize the OxlT translocation pathway. (3) Mutagenesis will also be used to identify functionally important residues, with emphasis on K355, which may be the lone charged residue in the OxlT hydrophobic core; K355 may form part of an anion binding center. (4) Concurrently, capitalizing on the stability of the OxlT/substrate complex and on recent success in protein purification, trials of 2-dimensional crystallization will be conducted. Together, this work will extend an understanding of OxlT from the level of cell physiology to that of molecular biology and biophysics. This project is about OxlT, a novel transport protein found in the soil bacterium, Ox alobacter formigenes. OxlT catalyzes the transmembrane exchange of two anions that differ in net charge, so that a membrane potential arises as one (divalent oxalate) moves inward while the other (monovalent formate) moves outward. Similarly, when inside the cell, oxalate is transformed into formate in a reaction that consumes a single proton. Overall, then, the metabolism of oxalate generates electrical and chemical (ph) gradients that comprise a "protonmotive force" which O. formigenes uses to power other membrane-associated events. Such proton-motive metabolic cycles appear to be widely spread in microorganisms. To understand these events at a molecular level this project concentrates on studies of OxlT, the antiport protein at the center of the process in O. formigenes. Initial studies will focus on the overall organization of OxlT in the cell membrane, using biochemical, genetic and immunological tools. Mutagenesis will then be used to change the molecule into one suitable for probing with reagents that may localize the substrate transport pathway and identify functionally important residues. Finally, trials of 2-dimensional crystallization will be conducted to capitalize on the stability of the OxlT-substrate complex. Together, such experiments should experiments should extend an understanding of OxlT from the level of cell physiology to that of molecular biology and biophysics.